The invention relates to mutant microorganisms, such as algae and heterokonts, having increased lipid productivity and methods of their use in producing lipids.
Many microorganisms such as algae, labyrinthulomycetes (“chytrids”), and oleaginous yeast induce lipid biosynthesis in response to nutrient stress, such as nitrogen starvation. Under conditions of nitrogen depletion, such microorganisms redirect compound biosynthesis from protein to storage lipids, typically triacylglyceride lipids (“TAG”). Because nitrogen depletion simultaneously decreases cell growth, optimal lipid biosynthesis is limited to a relatively short window before the cells become too metabolically impaired to maintain high levels of production.
Various attempts to improve lipid productivity by increasing lipid biosynthesis during nutrient replete growth have focused on manipulating genes encoding enzymes for nitrogen assimilation or lipid metabolism as well as genes encoding polypeptides involved in lipid storage. For example, US2014/0162330 discloses a Phaeodactylum tricornutum strain in which the nitrate reductase (NR) gene has been attenuated by RNAi-based knockdown; Trentacoste et al. ((2013) Proc. Natl. Acad. Sci. USA 110: 19748-19753) disclose diatoms transformed with an RNAi construct targeting the Thaps3_264297 gene predicted to be involved in lipid catabolism; and WO2011127118 discloses transformation of Chlamydomonas with genes encoding oleosins (lipid storage protein) as well as with genes encoding diacylglycerol transferase (DGAT) genes. Although in each case increased lipid production was asserted based on microscopy or staining with lipophilic dyes, no quantitation of lipid production by the manipulated cells was provided, nor was the relationship between biomass and lipid productivities over time determined.
Daboussi et al. 2014 (Nature Comm. 5:3881) report that disruption of the UGPase gene in Phaeodactylum triconornutum, which is believed to provide precursors to laminarin (a storage carbohydrate) synthesis, results in increased lipid accumulation. However, no biochemical data was shown to indicate that laminarin content was affected (or even present) and lipid and biomass productivities were not reported. Similarly, several groups have reported increases in lipid accumulation in Chlamydomonas starchless mutants (Wang et al. 2009 Eukaryotic Cell 8:1856-1868; Li et al. 2010 Metab Eng. 12:387-391) however, successive reports that actually measured lipid productivity concluded that these strains were impaired in growth when grown in phototrophic conditions (Siaut et al. 2011 BMC Biotechnol. 11:7; Davey et al. 2014 Eukaryot Cell 13:392-400). These reports concluded that the highest lipid productivities (measured as TAG per liter per day) were actually achieved by the wild-type parental strain.
WO 2011/097261 and US20120322157 report that a gene denoted “SN03” encoding an arrestin protein has a role in increasing lipid production under nutrient replete conditions when overexpressed in Chlamydomonas. However, overexpression of the SN03 gene was observed to result in the appearance of unidentified polar lipids, which were not quantified, and did not result in an increase in triglycerides (TAG). Another polypeptide identified as potentially regulating stress-induced lipid biosynthesis has been described by Boyle et al. ((2012) J. Biol. Chem. 287:15811-15825). Knockout of the NRR1 gene in Chlamydomonas encoding a “SQUAMOUSA” domain polypeptide resulted in a reduction of lipid biosynthesis with respect to wild type cells under nitrogen depletion; however, no mutants were obtained demonstrating increased lipid production. US 2010/0255550 suggests the overexpression of putative transcription factors (TF1, TF2, TF3, TF4, and TF5) in algal cells to increase lipid production, but no such strains are disclosed.
WO 2015/130832 and Ngan et al. (Nature Plants, 1:1507, 2015) report that overexpression of the PSR1 gene, a regulator of the phosphate starvation response, leads to cellular lipid accumulation in Chlamydomonas. Cells engineered to overexpress PSR1 are disclosed as having the large round phenotype of cells induced to produce lipid via nutrient starvation. Transformed cells were also observed to have higher amounts of cellular lipid with respect to control cells as visualized by electron microscopy and measured by staining with lipophilic fluorophores. However, increased lipid production by cultures on a volumetric basis was not demonstrated, nor was culture propagation, biomass accumulation, or overexpression of the PSR1 gene determined from the cultures in which lipid accumulation was observed. Conversely, growth rate of the PSR1-transformed cultures was demonstrated under conditions that were nonselective for the expression of the gene, and neither PSR1 gene expression level nor lipid production was assessed during the growth assay period.
Copending and commonly-owned U.S. Utility application Ser. No. 15/210,845 filed Jul. 14, 2016, entitled “Microorganisms Having Increased Lipid Productivity” discloses algal mutants having attenuated expression of a lipid regulator gene “ZnCys-2845” referred to herein as “LION1” mutants and their use in lipid production.